Cell lines producing blocking antibodies to human RANKL

ABSTRACT

Described herein are cell lines and methods for preparing antibodies that bind RANKL, including cell lines that produce blocking antibodies to human RANKL.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/214,914, filed Jun. 24, 2008, now U.S. Pat. No. 7,744,886, which isincorporated herein by reference in its entirety for all purposes, whichis a divisional of U.S. application Ser. No. 10/802,133, filed Mar. 16,2004, now U.S. Pat. No. 7,411,050, which is a continuation of U.S.application Ser. No. 09/865,363, filed May 25, 2001, now U.S. Pat. No.6,740,522, which is a divisional of U.S. application Ser. No. 09/577,780filed May 24, 2000, now U.S. Pat. No. 6,419,929, which is a divisionalof U.S. application Ser. No. 08/995,659 filed Dec. 22, 1997, now U.S.Pat. No. 6,242,213, which claims benefit of U.S. applications60/064,671, filed Oct. 14, 1997, 60/077,181 filed Mar. 7, 1997, and60/059,978 filed Dec. 23, 1996.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to the field of cytokines, andmore specifically to cytokine receptor/ligand pairs havingimmunoregulatory activity.

BACKGROUND OF THE INVENTION

Efficient functioning of the immune system requires a fine balancebetween cell proliferation and differentiation and cell death, to ensurethat the immune system is capable of reacting to foreign, but not selfantigens. Integral to the process of regulating the immune andinflammatory response are various members of the Tumor Necrosis Factor(TNF) Receptor/Nerve Growth Factor Receptor superfamily (Smith et al.,Science 248:1019; 1990). This family of receptors includes two differentTNF receptors (Type I and Type II; Smith et al., supra; and Schall etal., Cell 61:361, 1990), nerve growth factor receptor (Johnson et al.,Cell 47:545, 1986), B cell antigen CD40 (Stamenkovic et al., EMBO J.8:1403, 1989), CD27 (Camerini et al., J. Immunol. 147:3165, 1991), CD30(Durkop et al., Cell 68:421, 1992), T cell antigen OX40 (Mallett et al.,EMBO J. 9:1063, 1990), human Fas antigen (Itoh et al., Cell 66:233,1991), murine 4-1BB receptor (Kwon et al., Proc. Natl. Acad. Sci. USA86:1963, 1989) and a receptor referred to as Apoptosis-Inducing Receptor(AIR; U.S. Ser. No. 08/720,864, filed Oct. 4, 1996).

CD40 is a receptor present on B lymphocytes, epithelial cells and somecarcinoma cell lines that interacts with a ligand found on activated Tcells, CD40L (U.S. Ser. No. 08/249,189, filed May 24, 1994). Theinteraction of this ligand/receptor pair is essential for both thecellular and humoral immune response. Signal transduction via CD40 ismediated through the association of the cytoplasmic domain of thismolecule with members of the TNF receptor-associated factors (TRAFs;Baker and Reddy, Oncogene 12:1, 1996). It has recently been found thatmice that are defective in TRAF3 expression due to a targeted disruptionin the gene encoding TRAF3 appear normal at birth but developprogressive hypoglycemia and depletion of peripheral white cells, anddie by about ten days of age (Xu et al., Immunity 5:407, 1996). Theimmune responses of chimeric mice reconstituted with TRAF3^(−/−) fetalliver cells resemble those of CD40-deficient mice, although TRAF3^(−/−)B cells appear to be functionally normal.

The critical role of TRAF3 in signal transduction may be in itsinteraction with one of the other members of the TNF receptorsuperfamily, for example, CD30 or CD27, which are present on T cells.Alternatively, there may be other, as yet unidentified members of thisfamily of receptors that interact with TRAF3 and play an important rolein postnatal development as well as in the development of a competentimmune system. Identifying additional members of the TNF receptorsuperfamily would provide an additional means of regulating the immuneand inflammatory response, as well as potentially providing furtherinsight into post-natal development in mammals.

SUMMARY OF THE INVENTION

The present invention provides a counterstructure, or ligand, for anovel receptor referred to as RANK (for receptor activator of NF-κB),that is a member of the TNF superfamily. The ligand, which is referredto as RANKL, is a Type 2 transmembrane protein with an intracellulardomain of less than about 50 amino acids, a transmembrane domain and anextracellular domain of from about 240 to 250 amino acids. Similar toother members of the TNF family to which it belongs, RANKL has a‘spacer’ region between the transmembrane domain and the receptorbinding domain that is not necessary for receptor binding. Accordingly,soluble forms of RANKL can comprise the entire extracellular domain orfragments thereof that include the receptor binding region.

RANK is a Type I transmembrane protein having 616 amino acid residuesthat is a member of the TNFR superfamily, and interacts with TRAF3.Triggering of RANK by over-expression, co-expression of RANK andmembrane bound RANKL, or by soluble RANKL or agonistic antibodies toRANK, results in the upregulation of the transcription factor NF-κB, aubiquitous transcription factor that is most extensively utilized incells of the immune system.

These and other aspects of the present invention will become evidentupon reference to the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the influence of RANK.Fc and hRANKL on activated Tcell growth. Human peripheral blood T cells were cultured as describedin Example 12; viable T cell recovery was determined by triplicatetrypan blue countings.

FIG. 2 demonstrates that RANKL enhances DC allo-stimulatory capacity.Allogeneic T cells were incubated with varying numbers of irradiated DCcultured as described in Example 13. The cultures were pulsed with[³H]-thymidine and the cells harvested onto glass fiber sheets forcounting. Values represent the mean±standard deviation (SD) oftriplicate cultures.

DETAILED DESCRIPTION OF THE INVENTION

A novel partial cDNA insert with a predicted open reading frame havingsome similarity to CD40 was identified in a database containing sequenceinformation from cDNAs generated from human bone marrow-deriveddendritic cells (DC). The insert was used to hybridize to colony blotsgenerated from a DC cDNA library containing full-length cDNAs. Severalcolony hybridizations were performed, and two clones (SEQ ID NOs:1 and3) were isolated. SEQ ID NO:5 shows the nucleotide and amino acidsequence of a predicted full-length protein based on alignment of theoverlapping sequences of SEQ ID NOs:1 and 3.

RANK is a member of the TNF receptor superfamily; it most closelyresembles CD40 in the extracellular region. Similar to CD40, RANKassociates with TRAF2 and TRAF3 (as determined by co-immunoprecipitationassays substantially as described by Rothe et al., Cell 83:1243, 1995).TRAFs are critically important in the regulation of the immune andinflammatory response. Through their association with various members ofthe TNF receptor superfamily, a signal is transduced to a cell. Thatsignal results in the proliferation, differentiation or apoptosis of thecell, depending on which receptor(s) is/are triggered and which TRAF(s)associate with the receptor(s); different signals can be transduced to acell via coordination of various signaling events. Thus, a signaltransduced through one member of this family may be proliferative,differentiative or apoptotic, depending on other signals beingtransduced to the cell, and/or the state of differentiation of the cell.Such exquisite regulation of this proliferative/apoptotic pathway isnecessary to develop and maintain protection against pathogens;imbalances can result in autoimmune disease.

RANK is expressed on epithelial cells, some B cell lines, and onactivated T cells. However, its expression on activated T cells is late,about four days after activation. This time course of expressioncoincides with the expression of Fas, a known agent of apoptosis. RANKmay act as an anti-apoptotic signal, rescuing cells that express RANKfrom apoptosis as CD40 is known to do. Alternatively, RANK may confirman apoptotic signal under the appropriate circumstances, again similarto CD40. RANK and its ligand are likely to play an integral role inregulation of the immune and inflammatory response.

Moreover, the post-natal lethality of mice having a targeted disruptionof the TRAF3 gene demonstrates the importance of this molecule not onlyin the immune response but in development. The isolation of RANK, as aprotein that associates with TRAF3, and its ligand, RANKL, will allowfurther definition of this signaling pathway, and development ofdiagnostic and therapeutic modalities for use in the area of autoimmuneand/or inflammatory disease.

DNAs, Proteins and Analogs

The present invention provides isolated RANKL polypeptides and analogs(or muteins) thereof having an activity exhibited by the native molecule(i.e, RANKL muteins that bind specifically to a RANK expressed on cellsor immobilized on a surface or to RANKL-specific antibodies; solubleforms thereof that inhibit RANK ligand-induced signaling through RANK).Such proteins are substantially free of contaminating endogenousmaterials and, optionally, without associated native-patternglycosylation. Derivatives of RANKL within the scope of the inventionalso include various structural forms of the primary proteins whichretain biological activity. Due to the presence of ionizable amino andcarboxyl groups, for example, a RANKL protein may be in the form ofacidic or basic salts, or may be in neutral form. Individual amino acidresidues may also be modified by oxidation or reduction. The primaryamino acid structure may be modified by forming covalent or aggregativeconjugates with other chemical moieties, such as glycosyl groups,lipids, phosphate, acetyl groups and the like, or by creating amino acidsequence mutants. Covalent derivatives are prepared by linkingparticular functional groups to amino acid side chains or at the N- orC-termini.

Derivatives of RANKL may also be obtained by the action of cross-linkingagents, such as M-maleimidobenzoyl succinimide ester andN-hydroxysuccinimide, at cysteine and lysine residues. The inventiveproteins may also be covalently bound through reactive side groups tovarious insoluble substrates, such as cyanogen bromide-activated,bisoxirane-activated, carbonyldiimidazole-activated or tosyl-activatedagarose structures, or by adsorbing to polyolefin surfaces (with orwithout glutaraldehyde cross-linking). Once bound to a substrate, theproteins may be used to selectively bind (for purposes of assay orpurification) antibodies raised against the proteins or against otherproteins which are similar to RANKL, as well as other proteins that bindRANKL or homologs thereof.

Soluble forms of RANKL are also within the scope of the invention. Thenucleotide and predicted amino acid sequence of the RANKL is shown inSEQ ID Nos:11 and 13 (murine and human, respectively). Computer analysisindicated that the RANKL is a Type 2 transmembrane protein; murine RANKLcontains a predicted 48 amino acid intracellular domain, 21 amino acidtransmembrane domain and 247 amino acid extracellular domain, and humanRANKL contains a predicted 47 amino acid intracellular domain, 21 aminoacid transmembrane domain and 249 amino acid extracellular domain.

Soluble RANKL comprises a signal peptide and the extracellular domain ora fragment thereof. An exemplary signal peptide is that shown in SEQ IDNO:9; other signal (or leader) peptides are well-known in the art, andinclude that of murine Interleukin-7 or human growth hormone. RANKL issimilar to other members of the TNF family in having a region of aminoacids between the transmembrane domain and the receptor binding regionthat does not appear to be required for biological activity; this isreferred to as a ‘spacer’ region. Amino acid sequence alignmentindicates that the receptor binding region is from about amino acid 162of human RANKL to about amino acid 317 (corresponding to amino acid 139through 294 of murine RANKL, SEQ ID NO:11), beginning with an Alaresidue that is conserved among many members of the family (amino acid162 of SEQ ID NO:13).

Moreover, fragments of the extracellular domain will also providesoluble forms of RANKL. Those skilled in the art will recognize that theactual receptor binding region may be different than that predicted bycomputer analysis. Thus, the N-terminal amino acid of a soluble RANKL isexpected to be within about five amino acids on either side of theconserved Ala residue. Alternatively, all or a portion of the spacerregion may be included at the N-terminus of a soluble RANKL, as may beall or a portion of the transmembrane and/or intracellular domains,provided that the resulting soluble RANKL, is not membrane-associated.Accordingly, a soluble RANKL will have an N-terminal amino acid selectedfrom the group consisting of amino acids 1 through 162 of SEQ ID NO:13(1 though 139 of SEQ ID NO:11). Preferably, the amino terminal aminoacid is between amino acids 69 and 162 of SEQ ID NO:13 (human RANKL;amino acids 48 and 139 of SEQ ID NO:11). Similarly, the carboxy terminalamino acid can be between amino acid 313 and 317 of SEQ ID NO:13 (humanRANKL; corresponding to amino acids 290 through 294 of SEQ ID NO:11).Those skilled in the art can prepare these and additional soluble formsthrough routine experimentation.

Fragments can be prepared using known techniques to isolate a desiredportion of the extracellular region, and can be prepared, for example,by comparing the extracellular region with those of other members of theTNF family (of which RANKL is a member) and selecting forms similar tothose prepared for other family members. Alternatively, uniquerestriction sites or PCR techniques that are known in the art can beused to prepare numerous truncated forms which can be expressed andanalyzed for activity.

Other derivatives of the RANKL proteins within the scope of thisinvention include covalent or aggregative conjugates of the proteins ortheir fragments with other proteins or polypeptides, such as bysynthesis in recombinant culture as N-terminal or C-terminal fusions.For example, the conjugated peptide may be a signal (or leader)polypeptide sequence at the N-terminal region of the protein whichco-translationally or post-translationally directs transfer of theprotein from its site of synthesis to its site of function inside oroutside of the cell membrane or wall (e.g., the yeast α-factor leader).

Protein fusions can comprise peptides added to facilitate purificationor identification of RANKL proteins and homologs (e.g., poly-His). Theamino acid sequence of the inventive proteins can also be linked to anidentification peptide such as that described by Hopp et al.,Bio/Technology 6:1204 (1988). Such a highly antigenic peptide providesan epitope reversibly bound by a specific monoclonal antibody, enablingrapid assay and facile purification of expressed recombinant protein.The sequence of Hopp et al. is also specifically cleaved by bovinemucosal enterokinase, allowing removal of the peptide from the purifiedprotein. Fusion proteins capped with such peptides may also be resistantto intracellular degradation in E. coli.

Fusion proteins further comprise the amino acid sequence of a RANKLlinked to an immunoglobulin Fc region. An exemplary Fc region is a humanIgG₁ having a nucleotide an amino acid sequence set forth in SEQ IDNO:8. Fragments of an Fc region may also be used, as can Fc muteins. Forexample, certain residues within the hinge region of an Fc region arecritical for high affinity binding to FcγRO. Canfield and Morrison (J.Exp. Med. 173:1483; 1991) reported that Leu₍₂₃₄₎ and Leu₍₂₃₅₎ werecritical to high affinity binding of IgG₃ to FcγRI present on U937cells. Similar results were obtained by Lund et al. (J. Immunol.147:2657, 1991; Molecular Immunol. 29:53, 1991). Such mutations, aloneor in combination, can be made in an IgG₁ Fc region to decrease theaffinity of IgG₁ for FcR. Depending on the portion of the Fc regionused, a fusion protein may be expressed as a dimer, through formation ofinterchain disulfide bonds. If the fusion proteins are made with bothheavy and light chains of an antibody, it is possible to form a proteinoligomer with as many as four RANKL regions.

In another embodiment, RANKL proteins further comprise an oligomerizingpeptide such as a leucine zipper domain. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, 1988). Leucine zipper domain is a term used to refer to aconserved peptide domain present in these (and other) proteins, which isresponsible for dimerization of the proteins. The leucine zipper domain(also referred to herein as an oligomerizing, or oligomer-forming,domain) comprises a repetitive heptad repeat, with four or five leucineresidues interspersed with other amino acids. Examples of leucine zipperdomains are those found in the yeast transcription factor GCN4 and aheat-stable DNA-binding protein found in rat liver (C/EBP; Landschulz etal., Science 243:1681, 1989). Two nuclear transforming proteins, fos andjun, also exhibit leucine zipper domains, as does the gene product ofthe murine proto-oncogene, c-myc (Landschulz et al., Science 240:1759,1988). The products of the nuclear oncogenes fos and jun compriseleucine zipper domains preferentially form a heterodimer (O'Shea et al.,Science 245:646, 1989; Turner and Tjian, Science 243:1689, 1989). Theleucine zipper domain is necessary for biological activity (DNA binding)in these proteins.

The fusogenic proteins of several different viruses, includingparamyxovirus, coronavirus, measles virus and many retroviruses, alsopossess leucine zipper domains (Buckland and Wild, Nature 338:547, 1989;Britton, Nature 353:394, 1991; Delwart and Mosialos, AIDS Research andHuman Retroviruses 6:703, 1990). The leucine zipper domains in thesefusogenic viral proteins are near the transmembrane region of theproteins; it has been suggested that the leucine zipper domains couldcontribute to the oligomeric structure of the fusogenic proteins.Oligomerization of fusogenic viral proteins is involved in fusion poreformation (Spruce et al, Proc. Natl. Acad. Sci. U.S.A. 88:3523, 1991).Leucine zipper domains have also been recently reported to play a rolein oligomerization of heat-shock transcription factors (Rabindran etal., Science 259:230, 1993).

Leucine zipper domains fold as short, parallel coiled coils. (O'Shea etal., Science 254:539; 1991) The general architecture of the parallelcoiled coil has been well characterized, with a “knobs-into-holes”packing as proposed by Crick in 1953 (Acta Crystallogr. 6:689). Thedimer formed by a leucine zipper domain is stabilized by the heptadrepeat, designated (abcdefg)_(n) according to the notation of McLachlanand Stewart (J. Mol. Biol. 98:293; 1975), in which residues a and d aregenerally hydrophobic residues, with d being a leucine, which line up onthe same face of a helix. Oppositely-charged residues commonly occur atpositions g and e. Thus, in a parallel coiled coil formed from twohelical leucine zipper domains, the “knobs” formed by the hydrophobicside chains of the first helix are packed into the “holes” formedbetween the side chains of the second helix.

The leucine residues at position d contribute large hydrophobicstabilization energies, and are important for dimer formation (Krysteket al., Int. J. Peptide Res. 38:229, 1991). Lovejoy et al. recentlyreported the synthesis of a triple-stranded α-helical bundle in whichthe helices run up-up-down (Science 259:1288, 1993). Their studiesconfirmed that hydrophobic stabilization energy provides the maindriving force for the formation of coiled coils from helical monomers.These studies also indicate that electrostatic interactions contributeto the stoichiometry and geometry of coiled coils.

Several studies have indicated that conservative amino acids may besubstituted for individual leucine residues with minimal decrease in theability to dimerize; multiple changes, however, usually result in lossof this ability (Landschulz et al., Science 243:1681, 1989; Turner andTjian, Science 243:1689, 1989; Hu et al., Science 250:1400, 1990). vanHeekeren et al. reported that a number of different amino residues canbe substituted for the leucine residues in the leucine zipper domain ofGCN4, and further found that some GCN4 proteins containing two leucinesubstitutions were weakly active (Nucl. Acids Res. 20:3721, 1992).Mutation of the first and second heptadic leucines of the leucine zipperdomain of the measles virus fusion protein (MVF) did not affectsyncytium formation (a measure of virally-induced cell fusion); however,mutation of all four leucine residues prevented fusion completely(Buckland et al., J. Gen. Virol. 73:1703, 1992). None of the mutationsaffected the ability of MVF to form a tetramer.

Amino acid substitutions in the a and d residues of a synthetic peptiderepresenting the GCN4 leucine zipper domain have been found to changethe oligomerization properties of the leucine zipper domain (Alber,Sixth Symposium of the Protein Society, San Diego, Calif.). When allresidues at position a are changed to isoleucine, the leucine zipperstill forms a parallel dimer. When, in addition to this change, allleucine residues at position d are also changed to isoleucine, theresultant peptide spontaneously forms a trimeric parallel coiled coil insolution. Substituting all amino acids at position d with isoleucine andat position a with leucine results in a peptide that tetramerizes.Peptides containing these substitutions are still referred to as leucinezipper domains.

The present invention also includes RANKL with or without associatednative-pattern glycosylation. Proteins expressed in yeast or mammalianexpression systems, e.g., COS-7 cells, may be similar or slightlydifferent in molecular weight and glycosylation pattern than the nativemolecules, depending upon the expression system. Expression of DNAsencoding the inventive proteins in bacteria such as E. coli providesnon-glycosylated molecules. Functional mutant analogs of RANKL proteinhaving inactivated N-glycosylation sites can be produced byoligonucleotide synthesis and ligation or by site-specific mutagenesistechniques. These analog proteins can be produced in a homogeneous,reduced-carbohydrate form in good yield using yeast expression systems.N-glycosylation sites in eukaryotic proteins are characterized by theamino acid triplet Asn-A₁-Z, where A₁ is any amino acid except Pro, andZ is Ser or Thr. In this sequence, asparagine provides a side chainamino group for covalent attachment of carbohydrate. Such a site can beeliminated by substituting another amino acid for Asn or for residue Z,deleting Asn or Z, or inserting a non-Z amino acid between A₁ and Z, oran amino acid other than Asn between Asn and A₁.

RANKL protein derivatives may also be obtained by mutations of thenative RANKL or subunits thereof. A RANKL mutated protein, as referredto herein, is a polypeptide homologous to a native RANKL protein, butwhich has an amino acid sequence different from the native proteinbecause of one or a plurality of deletions, insertions or substitutions.The effect of any mutation made in a DNA encoding a mutated peptide maybe easily determined by analyzing the ability of the mutated peptide tobind its counterstructure in a specific manner. Moreover, activity ofRANKL analogs, muteins or derivatives can be determined by any of theassays described herein (for example, induction of NF-κB activation).

Analogs of the inventive proteins may be constructed by, for example,making various substitutions of residues or sequences or deletingterminal or internal residues or sequences not needed for biologicalactivity. For example, cysteine residues can be deleted or replaced withother amino acids to prevent formation of incorrect intramoleculardisulfide bridges upon renaturation. Other approaches to mutagenesisinvolve modification of adjacent dibasic amino acid residues to enhanceexpression in yeast systems in which KEX2 protease activity is present.

When a deletion or insertion strategy is adopted, the potential effectof the deletion or insertion on biological activity should beconsidered. Subunits of the inventive proteins may be constructed bydeleting terminal or internal residues or sequences. Soluble forms ofRANKL can be readily prepared and tested for their ability to induceNF-κB activation. Polypeptides corresponding to the cytoplasmic regions,and fragments thereof (for example, a death domain) can be prepared bysimilar techniques. Additional guidance as to the types of mutationsthat can be made is provided by a comparison of the sequence of RANKL toproteins that have similar structures, as well as by performingstructural analysis of the inventive RANKL proteins.

Generally, substitutions should be made conservatively; i.e., the mostpreferred substitute amino acids are those which do not affect thebiological activity of RANKL (i.e., ability of the inventive proteins tobind antibodies to the corresponding native protein in substantiallyequivalent a manner, the ability to bind the counterstructure insubstantially the same manner as the native protein, the ability toinduce a RANKL signal, or ability to induce NF-κB activation). Examplesof conservative substitutions include substitution of amino acidsoutside of the binding domain(s) (either ligand/receptor or antibodybinding areas for the extracellular domain, or regions that interactwith other, intracellular proteins for the cytoplasmic domain), andsubstitution of amino acids that do not alter the secondary and/ortertiary structure of the native protein. Additional examples includesubstituting one aliphatic residue for another, such as Ile, Val, Leu,or Ala for one another, or substitutions of one polar residue foranother, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Othersuch conservative substitutions, for example, substitutions of entireregions having similar hydrophobicity characteristics, are well known.

Mutations in nucleotide sequences constructed for expression of analogproteins or fragments thereof must, of course, preserve the readingframe phase of the coding sequences and preferably will not createcomplementary regions that could hybridize to produce secondary mRNAstructures such as loops or hairpins which would adversely affecttranslation of the mRNA.

Not all mutations in the nucleotide sequence which encodes a RANKLprotein or fragments thereof will be expressed in the final product, forexample, nucleotide substitutions may be made to enhance expression,primarily to avoid secondary structure loops in the transcribed mRNA(see EPA 75,444A, incorporated herein by reference), or to providecodons that are more readily translated by the selected host, e.g., thewell-known E. coli preference codons for E. coli expression.

Although a mutation site may be predetermined, it is not necessary thatthe nature of the mutation per se be predetermined. For example, inorder to select for optimum characteristics of mutants, randommutagenesis may be conducted and the expressed mutated proteins screenedfor the desired activity. Mutations can be introduced at particular lociby synthesizing oligonucleotides containing a mutant sequence, flankedby restriction sites enabling ligation to fragments of the nativesequence. Following ligation, the resulting reconstructed sequenceencodes an analog having the desired amino acid insertion, substitution,or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981); andU.S. Pat. Nos. 4,518,584 and 4,737,462 disclose suitable techniques, andare incorporated by reference herein.

Additional embodiments of the inventive proteins include RANKLpolypeptides encoded by DNAs capable of hybridizing to the DNAS of SEQID NO:10 or 12 under moderately stringent conditions (prewashingsolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridizationconditions of 50° C., 5×SSC, overnight) to the DNA sequences encodingRANKL, or more preferably under stringent conditions (for example,hybridization in 6×SSC at 63° C. overnight; washing in 3×SSC at 55° C.),and other sequences which are degenerate to those which encode theRANKL. In one embodiment, RANKL polypeptides are at least about 70%identical in amino acid sequence to the amino acid sequence of nativeRANKL protein as set forth in SEQ ID NOs:10 and 12. In a preferredembodiment, RANKL polypeptides are at least about 80% identical in aminoacid sequence to the native form of RANKL; most preferred polypeptidesare those that are at least about 90% identical to native RANKL.

Percent identity may be determined using a computer program, forexample, the GAP computer program described by Devereux et al. (Nucl.Acids Res. 12:387, 1984) and available from the University of WisconsinGenetics Computer Group (UWGCG). For fragments derived from the RANKLprotein, the identity is calculated based on that portion of the RANKLprotein that is present in the fragment

The biological activity of RANKL analogs or muteins can be determined bytesting the ability of the analogs or muteins to induce a signal throughRANK, for example, activation of transcription as described in theExamples herein. Alternatively, suitable assays, for example, an enzymeimmunoassay or a dot blot, employing an antibody that binds nativeRANKL, or a soluble form of RANK, can be used to assess the activity ofRANKL analogs or muteins. Suitable assays also include, for example,assays that measure the ability of a RANKL peptide or mutein to bindcells expressing RANK, and/or the biological effects thereon. Suchmethods are well known in the art.

Fragments of the RANKL nucleotide sequences are also useful. In oneembodiment, such fragments comprise at least about 17 consecutivenucleotides, preferably at least about 25 nucleotides, more preferablyat least 30 consecutive nucleotides, of the RANKL DNA disclosed herein.DNA and RNA, complements of such fragments are provided herein, alongwith both single-stranded and double-stranded forms of the RANKL DNAs ofSEQ ID NOs:10 and 12, and those encoding the aforementionedpolypeptides. A fragment of RANKL DNA generally comprises at least about17 nucleotides, preferably from about 17 to about 30 nucleotides. Suchnucleic acid fragments (for example, a probe corresponding to theextracellular domain of RANKL) are used as a probe or as primers in apolymerase chain reaction (PCR).

The probes also find use in detecting the presence of RANKL nucleicacids in in vitro assays and in such procedures as Northern and Southernblots. Cell types expressing RANKL can be identified as well. Suchprocedures are well known, and the skilled artisan can choose a probe ofsuitable length, depending on the particular intended application. ForPCR, 5′ and 3′ primers corresponding to the termini of a desired RANKLDNA sequence are employed to amplify that sequence, using conventionaltechniques.

Other useful fragments of the RANKL nucleic acids are antisense or senseoligonucleotides comprising a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target RANKL mRNA (sense) orRANKL DNA (antisense) sequences. The ability to create an antisense or asense oligonucleotide, based upon a cDNA sequence for a given protein isdescribed in, for example, Stein and Cohen, Cancer Res. 48:2659, 1988and van der Krol et al., BioTechniques 6:958, 1988.

Uses of DNAs, Proteins and Analogs

The RANKL DNAs, proteins and analogs described herein will have numeroususes, including the preparation of pharmaceutical compositions. Forexample, soluble forms of RANKL will be useful to transduce signal viaRANK. RANKL compositions (both protein and DNAs) will also be useful indevelopment of antibodies to RANKL, both those that inhibit binding toRANK and those that do not. The inventive DNAs are useful for theexpression of recombinant proteins, and as probes for analysis (eitherquantitative or qualitative) of the presence or distribution of RANKLtranscripts.

The inventive proteins will also be useful in preparing kits that areused to detect soluble RANK or RANKL, or monitor RANK-related activity,for example, in patient specimens. RANKL proteins will also find uses inmonitoring RANK-related activity in other samples or compositions, as isnecessary when screening for antagonists or mimetics of this activity(for example, peptides or small molecules that inhibit or mimic,respectively, the interaction). A variety of assay formats are useful insuch kits, including (but not limited to) ELISA, dot blot, solid phasebinding assays (such as those using a biosensor), rapid format assaysand bioassays.

The purified RANKL according to the invention will facilitate thediscovery of inhibitors of RANK, and thus, inhibitors of an inflammatoryresponse (via inhibition of NF-κB activation). The use of a purifiedRANKL polypeptide in the screening for potential inhibitors is importantand can virtually eliminate the possibility of interfering reactionswith contaminants. Such a screening assay can utilize either theextracellular domain of RANKL, or a fragment thereof. Detecting theinhibiting activity of a molecule would typically involve use of asoluble form of RANKL derived from the extracellular domain in ascreening assay to detect molecules capable of binding RANK andinhibiting binding of the RANKL.

In addition, RANKL polypeptides can also be used for structure-baseddesign of RANKL-inhibitors. Such structure-based design is also known as“rational drug design.” The RANKL polypeptides can bethree-dimensionally analyzed by, for example, X-ray crystallography,nuclear magnetic resonance or homology modeling, all of which arewell-known methods. The use of RANKL structural information in molecularmodeling software systems to assist in inhibitor design is alsoencompassed by the invention. Such computer-assisted modeling and drugdesign may utilize information such as chemical conformational analysis,electrostatic potential of the molecules, protein folding, etc. Aparticular method of the invention comprises analyzing the threedimensional structure of RANKL for likely binding sites of substrates,synthesizing a new molecule that incorporates a predictive reactivesite, and assaying the new molecule as described above.

Moreover, as shown in the Examples herein, soluble forms of RANKL willbe useful to induce maturation of dendritic cells (DC), and to enhancetheir allo-stimulatory capacity. Accordingly, RANKL proteins will beuseful in augmenting an immune response, and can be used for thesepurposes either ex vivo (i.e., in obtaining cells such as DC from anindividual, exposing them to antigen and cytokines ex vivo, andre-administering them to the individual) or in vivo (i.e., as a vaccineadjuvant that will augment humoral and/or cellular immunity). RANKL willalso be useful promoting viability of T cells in the presence of TGFβ,which will also be helpful in regulating an immune response.

Expression of Recombinant RANKL

The proteins of the present invention are preferably produced byrecombinant DNA methods by inserting a DNA sequence encoding RANKLprotein or an analog thereof into a recombinant expression vector andexpressing the DNA sequence in a recombinant expression system underconditions promoting expression. DNA sequences encoding the proteinsprovided by this invention can be assembled from cDNA fragments andshort oligonucleotide linkers, or from a series of oligonucleotides, toprovide a synthetic gene which is capable of being inserted in arecombinant expression vector and expressed in a recombinanttranscriptional unit.

Recombinant expression vectors include synthetic or cDNA-derived DNAfragments encoding RANKL, or homologs, muteins or bioequivalent analogsthereof, operably linked to suitable transcriptional or translationalregulatory elements derived from mammalian, microbial, viral or insectgenes. Such regulatory elements include a transcriptional promoter, anoptional operator sequence to control transcription, a sequence encodingsuitable mRNA ribosomal binding sites, and sequences which control thetermination of transcription and translation, as described in detailbelow. The ability to replicate in a host, usually conferred by anorigin of replication, and a selection gene to facilitate recognition oftransformants may additionally be incorporated.

DNA regions are operably linked when they are functionally related toeach other. For example, DNA for a signal peptide (secretory leader) isoperably linked to DNA for a polypeptide if it is expressed as aprecursor which participates in the secretion of the polypeptide; apromoter is operably linked to a coding sequence if it controls thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to permittranslation. Generally, operably linked means contiguous and, in thecase of secretory leaders, contiguous and in reading frame. DNAsequences encoding RANKL, or homologs or analogs thereof which are to beexpressed in a microorganism will preferably contain no introns thatcould prematurely terminate transcription of DNA into mRNA.

Useful expression vectors for bacterial use can comprise a selectablemarker and bacterial origin of replication derived from commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017). Such commercial vectors include, forexample, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1(Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sectionsare combined with an appropriate promoter and the structural sequence tobe expressed. E. coli is typically transformed using derivatives ofpBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene2:95, 1977). pBR322 contains genes for ampicillin and tetracyclineresistance and thus provides simple means for identifying transformedcells.

Promoters commonly used in recombinant microbial expression vectorsinclude the β-lactamase (penicillinase) and lactose promoter system(Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544,1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. AcidsRes. 8:4057, 1980; and EPA 36,776) and tac promoter (Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412,1982). A particularly useful bacterial expression system employs thephage λ P_(L) promoter and cI857ts thermolabile repressor. Plasmidvectors available from the American Type Culture Collection whichincorporate derivatives of the λ P_(L) promoter include plasmid pHUB2,resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E.coli RR1 (ATCC 53082).

Suitable promoter sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Suitable vectorsand promoters for use in yeast expression are further described in R.Hitzeman et al., EPA 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pBR322for selection and replication in E. coli (Ampr gene and origin ofreplication) and yeast DNA sequences including a glucose-repressibleADH2 promoter and α-factor secretion leader. The ADH2 promoter has beendescribed by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier etal. (Nature 300:724, 1982). The yeast α-factor leader, which directssecretion of heterologous proteins, can be inserted between the promoterand the structural gene to be expressed. See, e.g., Kurjan et al., Cell30:933, 1982; and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330,1984. The leader sequence may be modified to contain, near its 3′ end,one or more useful restriction sites to facilitate fusion of the leadersequence to foreign genes.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells may be provided byviral sources. For example, commonly used promoters and enhancers arederived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and humancytomegalovirus. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites may be used to provide the other genetic elementsrequired for expression of a heterologous DNA sequence. The early andlate promoters are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication (Fiers et al., Nature 273:113, 1978). Smaller or largerSV40 fragments may also be used, provided the approximately 250 bpsequence extending from the Hind III site toward the BglI site locatedin the viral origin of replication is included. Further, viral genomicpromoter, control and/or signal sequences may be utilized, provided suchcontrol sequences are compatible with the host cell chosen. Exemplaryvectors can be constructed as disclosed by Okayama and Berg (Mol. Cell.Biol. 3:280, 1983).

A useful system for stable high level expression of mammalian receptorcDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986). A preferred eukaryotic vector for expression of RANKL DNA isreferred to as pDC406 (McMahan et al., EMBO J. 10:2821, 1991), andincludes regulatory sequences derived from SV40, human immunodeficiencyvirus (HIV), and Epstein-Barr virus (EBV). Other preferred vectorsinclude pDC409 and pDC410, which are derived from pDC406. pDC410 wasderived from pDC406 bp substituting the EBV origin of replication withsequences encoding the SV40 large T antigen. pDC409 differs from pDC406in that a Bgl II restriction site outside of the multiple cloning sitehas been deleted, making the Bgl II site within the multiple cloningsite unique.

A useful cell line that allows for episomal replication of expressionvectors, such as pDC406 and pDC409, which contain the EBV origin ofreplication, is CV-1/EBNA (ATCC CRL 10478). The CV-1/EBNA cell line wasderived by transfection of the CV-1 cell line with a gene encodingEpstein-Barr virus nuclear antigen-1 (EBNA-1) and constitutively expressEBNA-1 driven from human CMV immediate-early enhancer/promoter.

Host Cells

Transformed host cells are cells which have been transformed ortransfected with expression vectors constructed using recombinant DNAtechniques and which contain sequences encoding the proteins of thepresent invention. Transformed host cells may express the desiredprotein (RANKL, or homologs or analogs thereof), but host cellstransformed for purposes of cloning or amplifying the inventive DNA donot need to express the protein. Expressed proteins will preferably besecreted into the culture supernatant, depending on the DNA selected,but may be deposited in the cell membrane.

Suitable host cells for expression of proteins include prokaryotes,yeast or higher eukaryotic cells under the control of appropriatepromoters. Prokaryotes include gram negative or gram positive organisms,for example E. coli or Bacillus spp. Higher eukaryotic cells includeestablished cell lines of mammalian origin as described below. Cell-freetranslation systems could also be employed to produce proteins usingRNAs derived from the DNA constructs disclosed herein. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described by Pouwels et al. (CloningVectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevantdisclosure of which is hereby incorporated by reference.

Prokaryotic expression hosts may be used for expression of RANKL, orhomologs or analogs thereof that do not require extensive proteolyticand disulfide processing. Prokaryotic expression vectors generallycomprise one or more phenotypic selectable markers, for example a geneencoding proteins conferring antibiotic resistance or supplying anautotrophic requirement, and an origin of replication recognized by thehost to ensure amplification within the host. Suitable prokaryotic hostsfor transformation include E. coli, Bacillus subtilis, Salmonellatyphimurium, and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

Recombinant RANKL may also be expressed in yeast hosts, preferably fromthe Saccharomyces species, such as S. cerevisiae. Yeast of other genera,such as Pichia or Kluyveromyces may also be employed. Yeast vectors willgenerally contain an origin of replication from the 2μ yeast plasmid oran autonomously replicating sequence (ARS), promoter, DNA encoding theprotein, sequences for polyadenylation and transcription termination anda selection gene. Preferably, yeast vectors will include an origin ofreplication and selectable marker permitting transformation of bothyeast and E. coli, e.g., the ampicillin resistance gene of E. coli andS. cerevisiae trp1 gene, which provides a selection marker for a mutantstrain of yeast lacking the ability to grow in tryptophan, and apromoter derived from a highly expressed yeast gene to inducetranscription of a structural sequence downstream. The presence of thetrp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan.

Suitable yeast transformation protocols are known to those of skill inthe art; an exemplary technique is described by Hinnen et al., Proc.Natl. Acad. Sci. USA 75:1929, 1978, selecting for Trp⁺ transformants ina selective medium consisting of 0.67% yeast nitrogen base, 0.5%casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/ml uracil. Hoststrains transformed by vectors comprising the ADH2 promoter may be grownfor expression in a rich medium consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs upon exhaustion ofmedium glucose. Crude yeast supernatants are harvested by filtration andheld at 4° C. prior to further purification.

Various mammalian or insect cell culture systems can be employed toexpress recombinant protein. Baculovirus systems for production ofheterologous proteins in insect cells are reviewed by Luckow andSummers, Bio/Technology 6:47 (1988). Examples of suitable mammalian hostcell lines include the COS-7 lines of monkey kidney cells, described byGluzman (Cell 23:175, 1981), and other cell lines capable of expressingan appropriate vector including, for example, CV-1/EBNA (ATCC CRL10478), L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHKcell lines. Mammalian expression vectors may comprise nontranscribedelements such as an origin of replication, a suitable promoter andenhancer linked to the gene to be expressed, and other 5′ or 3′ flankingnontranscribed sequences, and 5′ or 3′ nontranslated sequences, such asnecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, and transcriptional termination sequences.

Purification of Recombinant RANKL

Purified RANKL, and homologs or analogs thereof are prepared byculturing suitable host/vector systems to express the recombinanttranslation products of the DNAs of the present invention, which arethen purified from culture media or cell extracts. For example,supernatants from systems which secrete recombinant protein into culturemedia can be first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit.

Following the concentration step, the concentrate can be applied to asuitable purification matrix. For example, a suitable affinity matrixcan comprise a counter structure protein or lectin or antibody moleculebound to a suitable support. Alternatively, an anion exchange resin canbe employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.Gel filtration chromatography also provides a means of purifying theinventive proteins.

Affinity chromatography is a particularly preferred method of purifyingRANKL and homologs thereof. For example, a RANKL expressed as a fusionprotein comprising an immunoglobulin Fc region can be purified usingProtein A or Protein G affinity chromatography. Moreover, a RANKLprotein comprising an oligomerizing zipper domain may be purified on aresin comprising an antibody specific to the oligomerizing zipperdomain. Monoclonal antibodies against the RANKL protein may also beuseful in affinity chromatography purification, by utilizing methodsthat are well-known in the art. A ligand may also be used to prepare anaffinity matrix for affinity purification of RANKL.

Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a RANKL composition. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture is usually isolated byinitial extraction from cell pellets, followed by one or moreconcentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. Finally, high performance liquid chromatography(HPLC) can be employed for final purification steps. Microbial cellsemployed in expression of recombinant protein can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Fermentation of yeast which express the inventive protein as a secretedprotein greatly simplifies purification. Secreted recombinant proteinresulting from a large-scale fermentation can be purified by methodsanalogous to those disclosed by Urdal et al. (J. Chromatog. 296:171,1984). This reference describes two sequential, reversed-phase HPLCsteps for purification of recombinant human GM-CSF on a preparative HPLCcolumn.

Protein synthesized in recombinant culture is characterized by thepresence of cell components, including proteins, in amounts and of acharacter which depend upon the purification steps taken to recover theinventive protein from the culture. These components ordinarily will beof yeast, prokaryotic or non-human higher eukaryotic origin andpreferably are present in innocuous contaminant quantities, on the orderof less than about 1 percent by weight. Further, recombinant cellculture enables the production of the inventive proteins free of otherproteins which may be normally associated with the proteins as they arefound in nature in the species of origin.

Uses and Administration of RANKL Compositions

The present invention provides methods of using therapeutic compositionscomprising an effective amount of a protein and a suitable diluent andcarrier, and methods for regulating an immune or inflammatory response.The use of RANKL in conjunction with soluble cytokine receptors orcytokines, or other immunoregulatory molecules is also contemplated.

For therapeutic use, purified protein is administered to a patient,preferably a human, for treatment in a manner appropriate to theindication. Thus, for example, RANKL protein compositions administeredto regulate immune function can be given by bolus injection, continuousinfusion, sustained release from implants, or other suitable technique.Typically, a therapeutic agent will be administered in the form of acomposition comprising purified RANKL, in conjunction withphysiologically acceptable carriers, excipients or diluents. Suchcarriers will be nontoxic to recipients at the dosages andconcentrations employed.

Ordinarily, the preparation of such protein compositions entailscombining the inventive protein with buffers, antioxidants such asascorbic acid, low molecular weight (less than about 10 residues)polypeptides, proteins, amino acids, carbohydrates including glucose,sucrose or dextrins, chelating agents such as EDTA, glutathione andother stabilizers and excipients. Neutral buffered saline or salinemixed with conspecific serum albumin are exemplary appropriate diluents.Preferably, product is formulated as a lyophilizate using appropriateexcipient solutions (e.g., sucrose) as diluents. Appropriate dosages canbe determined in trials. The amount and frequency of administration willdepend, of course, on such factors as the nature and severity of theindication being treated, the desired response, the condition of thepatient, and so forth.

As shown herein, RANKL has beneficial effects on various cells importantin the immune system. Accordingly, RANKL may be adminstered to anindividual as a vaccine adjuvant, or as a therapeutic agent toupregulate an immune resposne, for example, ininfectious disease.Moreover, NF-κB has been found to play a protective role in preventingapoptotic death of cells induced by TNF-α or chemotherapy. Accordingly,agonists of RANK (i.e., RANKL and agonistic antibodies) will be usefulin protecting RANK-expressing cells from the negative effects ofchemotherapy or the presence of high levels of TNF-α such as occur insepsis (see, i.e., Barinaga, Science 274″ 41724, 1996, and the articlesby Beg and Baltimore and Wang et al., pages 782 and 784 of that sameissue of Science).

The following examples are offered by way of illustration, and not byway of limitation. Those skilled in the art will recognize thatvariations of the invention embodied in the examples can be made,especially in light of the teachings of the various references citedherein, the disclosures of which are incorporated by reference.

Example 1

The example describes the identification and isolation of a DNA encodinga novel member of the TNF receptor superfamily. A partial cDNA insertwith a predicted open reading frame having some similarity to CD40 (acell-surface antigen present on the surface of both normal andneoplastic human B cells that has been shown to play an important rolein B-cell proliferation and differentiation; Stamenkovic et al., EMBO J.8:1403, 1989), was identified in a database containing sequenceinformation from cDNAs generated from human bone marrow-deriveddendritic cells (DC). The insert was excised from the vector byrestriction endonuclease digestion, gel purified. labeled with ³²P, andused to hybridize to colony blots generated from a DC cDNA librarycontaining larger cDNA inserts using high stringency hybridization andwashing techniques (hybridization in 5×SSC, 50% formamide at 42° C.overnight, washing in 0.5×SSC at 63° C.); other suitable high stringencyconditions are disclosed in Sambrook et al. in Molecular Cloning: ALaboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.; 1989), 9.52-9.55. Initial experiments yielded a clonereferred to as 9D-8A (SEQ ID NO:1); subsequent analysis indicated thatthis clone contained all but the extreme 5′ end of a novel cDNA, withpredicted intron sequence at the extreme 5′ end (nucleotides 1-92 of SEQID NO:1). Additional colony hybridizations were performed, and a secondclone was isolated. The second clone, referred to as 9D-15C (SEQ IDNO:3), contained the 5′ end without intron interruption but not the full3′ end. SEQ ID NO:5 shows the nucleotide and amino acid sequence of apredicted full-length protein based on alignment of the overlappingsequences of SEQ ID NOs:1 and 3.

The encoded protein was designated RANK, for receptor activator ofNF-κB. The cDNA encodes a predicted Type 1 transmembrane protein having616 amino acid residues, with a predicted 24 amino acid signal sequence(the computer predicted cleavage site is after Leu24), a 188 amino acidextracellular domain, a 21 amino acid transmembrane domain, and a 383amino acid cytoplasmic tail. The extracellular region of RANK displayedsignificant amino acid homology (38.5% identity, 52.3% similarity) toCD40. A cloning vector (pBluescriptSK⁻) containing human RANK sequence,designated pBluescript:huRANK (in E. coli DH10B), was deposited with theAmerican Type Culture Collection, Rockville, Md. (ATCC) on Dec. 20,1996, under terms of the Budapest Treaty, and given accession number98285.

Example 2

This example describes construction of a RANK DNA construct to express aRANK/Fc fusion protein. A soluble form of RANK fused to the Fc region ofhuman IgG₁ was constructed in the mammalian expression vector pDC409(U.S. Ser. No. 08/571,579). This expression vector encodes the leadersequence of the Cytomegalovirus (CMV) open reading frame R27080 (SEQ IDNO:9), followed by amino acids 33-213 of RANK, followed by a mutatedform of the constant domain of human IgG₁ that exhibits reduced affinityfor Fc receptors (SEQ ID NO:8; for the fusion protein, the Fc portion ofthe construct consisted of Arg3 through Lys232). An alternativeexpression vector encompassing amino acids 1-213 of RANK (using thenative leader sequence) followed by the IgG₁ mutein was also prepared.Both expression vectors were found to induce high levels of expressionof the RANK/Fc fusion protein in transfected cells.

To obtain RANK/Fc protein, a RANK/Fc expression plasmid is transfectedinto CV-1/EBNA cells, and supernatants are collected for about one week.The RANK/Fc fusion protein is purified by means well-known in the artfor purification of Fc fusion proteins, for example, by protein Asepharose column chromatography according to manufacturer'srecommendations (i.e., Pharmacia, Uppsala, Sweden). SDS-polyacrylamidegel electrophoresis analysis indicted that the purified RANK/Fc proteinmigrated with a molecular weight of ˜55 kDa in the presence of areducing agent, and at a molecular weight of ˜110 kDa in the absence ofa reducing agent.

N-terminal amino acid sequencing of the purified protein made using theCMV R27080 leader showed 60% cleavage after Ala20, 20% cleavage afterPro22 and 20% cleavage after Arg28 (which is the Furin cleavage site;amino acid residues are relative to SEQ ID NO:9); N-terminal amino acidanalysis of the fusion protein expressed with the native leader showedcleavage predominantly after Gln25 (80% after Gln25 and 20% after Arg23;amino acid residues are relative to SEQ ID NO:6, full-length RANK). Bothfusion proteins were able to bind a ligand for RANK is a specific manner(i.e., they bound to the surface of various cell lines such as a murinethymoma cell line, EL4), indicating that the presence of additionalamino acids at the N-terminus of RANK does not interfere with itsability to bind RANKL. Moreover, the construct comprising the CMV leaderencoded RANK beginning at amino acid 33; thus, a RANK peptide having anN-terminus at an amino acid between Arg23 and Pro33, inclusive, isexpected to be able to bind a ligand for RANK in a specific manner.

Other members of the TNF receptor superfamily have a region of aminoacids between the transmembrane domain and the ligand binding domainthat is referred to as a ‘spacer’ region, which is not necessary forligand binding. In RANK, the amino acids between 196 and 213 arepredicted to form such a spacer region. Accordingly, a soluble form ofRANK that terminates with an amino acid in this region is expected toretain the ability to bind a ligand for RANK in a specific manner.Preferred C-terminal amino acids for soluble RANK peptides are selectedfrom the group consisting of amino acids 213 and 196 of SEQ ID NO:6,although other amino acids in the spacer region may be utilized as aC-terminus.

Example 3

This example illustrates the preparation of monoclonal antibodiesagainst RANK. Preparations of purified recombinant RANK, for example, ortransfected cells expressing high levels of RANK, are employed togenerate monoclonal antibodies against RANK using conventionaltechniques, such as those disclosed in U.S. Pat. No. 4,411,993. DNAencoding RANK can also be used as an immunogen, for example, as reviewedby Pardoll and Beckerleg in Immunity 3:165, 1995. Such antibodies arelikely to be useful in interfering with RANK-induced signaling(antagonistic or blocking antibodies) or in inducing a signal bycross-linking RANK (agonistic antibodies), as components of diagnosticor research assays for RANK or RANK activity, or in affinitypurification of RANK.

To immunize rodents, RANK immunogen is emulsified in an adjuvant (suchas complete or incomplete Freund's adjuvant, alum, or another adjuvant,such as Ribi adjuvant R700 (Ribi, Hamilton, Mont.), and injected inamounts ranging from 10-100 μg subcutaneously into a selected rodent,for example, BALB/c mice or Lewis rats. DNA may be given intradermally(Raz et al., Proc. Natl. Acad. Sci. USA 91:9519, 1994) or intamuscularly(Wang et al., Proc. Natl. Acad. Sci. USA 90:4156, 1993); saline has beenfound to be a suitable diluent for DNA-based antigens. Ten days to threeweeks days later, the immunized animals are boosted with additionalimmunogen and periodically boosted thereafter on a weekly, biweekly orevery third week immunization schedule.

Serum samples are periodically taken by retro-orbital bleeding ortail-tip excision for testing by dot-blot assay (antibody sandwich),ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or othersuitable assays, including FACS analysis. Following detection of anappropriate antibody titer, positive animals are given an intravenousinjection of antigen in saline. Three to four days later, the animalsare sacrificed, splenocytes harvested, and fused to a murine myelomacell line (e.g., NS1 or preferably Ag 8.653 [ATCC CRL 1580]). Hybridomacell lines generated by this procedure are plated in multiple microtiterplates in a selective medium (for example, one containing hypoxanthine,aminopterin, and thymidine, or HAT) to inhibit proliferation ofnon-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocytehybrids.

Hybridoma clones thus generated can be screened by ELISA for reactivitywith RANK, for example, by adaptations of the techniques disclosed byEngvall et al., Immunochem. 8:871 (1971) and in U.S. Pat. No. 4,703,004.A preferred screening technique is the antibody capture techniquedescribed by Beckman et al., J. Immunol. 144:4212 (1990). Positiveclones are then injected into the peritoneal cavities of syngeneicrodents to produce ascites containing high concentrations (>1 mg/ml) ofanti-RANK monoclonal antibody. The resulting monoclonal antibody can bepurified by ammonium sulfate precipitation followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can also be used, as canaffinity chromatography based upon binding to RANK protein.

Monoclonal antibodies were generated using RANK/Fc fusion protein as theimmunogen. These reagents were screened to confirm reactivity againstthe RANK protein. Using the methods described herein to monitor theactivity of the mAbs, both blocking (i.e., antibodies that bind RANK andinhibit binding of a ligand to RANK) and non-blocking (i.e., antibodiesthat bind RANK and do not inhibit ligand binding) were isolated.

Example 4

This example illustrates the induction of NF-κB activity by RANK in293/EBNA cells (cell line was derived by transfection of the 293 cellline with a gene encoding Epstein-Barr virus nuclear antigen-1 (EBNA-1)that constitutively express EBNA-1 driven from human CMV immediate-earlyenhancer/promoter). Activation of NF-κB activity was measured in293/EBNA cells essentially as described by Yao et al. (Immunity 3:811,1995). Nuclear extracts were prepared and analyzed for NF-κB activity bya gel retardation assay using a 25 base pair oligonucleotide spanningthe NF-κB binding sites. Two million cells were seeded into 10 cm dishestwo days prior to DNA transfection and cultured in DMEM-F12 mediacontaining 2.5% FBS (fetal bovine serum). DNA transfections wereperformed as described herein for the IL-8 promoter/reporter assays.

Nuclear extracts were prepared by solubilization of isolated nuclei with400 mM NaCl (Yao et al., supra). Oligonucleotides containing an NF-κBbinding site were annealed and endlabeled with ³²P using T4 DNApolynucleotide kinase. Mobility shift reactions contained 10 μg ofnuclear extract, 4 μg of poly(dI-dC) and 15,000 cpm labeleddouble-stranded oligonucleotide and incubated at room temperature for 20minutes. Resulting protein-DNA complexes were resolved on a 6% nativepolyacrylamide gel in 0.25× Tris-borate-EDTA buffer.

Overexpression of RANK resulted in induction of NF-κB activity as shownby an appropriate shift in the mobility of the radioactive probe on thegel. Similar results were observed when RANK was triggered by a ligandthat binds RANK and transduces a signal to cells expressing the receptor(i.e., by co-transfecting cells with human RANK and murine RANKL DNA;see Example 7 below), and would be expected to occur when triggering isdone with agonistic antibodies.

Example 5

This example describes a gene promoter/reporter system based on thehuman Interleukin-8 (IL-8) promoter used to analyze the activation ofgene transcription in vivo. The induction of human IL-8 genetranscription by the cytokines Interleukin-1 (IL-1) or tumor necrosisfactor-alpha (TNF-α) is known to be dependent upon intact NF-κB andNF-IL-6 transcription factor binding sites. Fusion of thecytokine-responsive IL-8 promoter with a cDNA encoding the murine IL-4receptor (mIL-4R) allows measurement of promoter activation by detectionof the heterologous reporter protein (mIL-4R) on the cell surface oftransfected cells.

Human kidney epithelial cells (293/EBNA) are transfected (via theDEAE/DEXTRAN method) with plasmids encoding: 1). the reporter/promoterconstruct (referred to as pIL-8rep), and 2). the cDNA(s) of interest.DNA concentrations are always kept constant by the addition of emptyvector DNA. The 293/EBNA cells are plated at a density of 2.5×10⁴cells/ml (3 ml/well) in a 6 well plate and incubated for two days priorto transfection. Two days after transfection, the mIL-4 receptor isdetected by a radioimmunoassay (RIA) described below.

In one such experiment, the 293/EBNA cells were co-transfected with DNAencoding RANK and with DNA encoding RANKL (see Example 7 below).Co-expression of this receptor and its counterstructure by cells resultsin activation of the signaling process of RANK. For such co-transfectionstudies, the DNA concentration/well for the DEAE transfection were asfollows: 40 ng of pIL-8rep [pBluescriptSK⁻ vector (Stratagene)]; 0.4 ngCD40 (DNA encoding CD40, a control receptor; pCDM8 vector); 0.4 ng RANK(DNA encoding RANK; pDC409 vector), and either 1-50 ng CD40L (DNAencoding the ligand for CD40, which acts as a positive control whenco-transfected with CD40 and as a negative control when co-transfectedwith RANK; in pDC304) or RANKL (DNA encoding a ligand for RANK; inpDC406). Similar experiments can be done using soluble RANKL oragonistic antibodies to RANK to trigger cells transfected with RANK.

For the mIL-4R-specific RIA, a monoclonal antibody reactive with mIL-4Ris labeled with ¹²⁵I via a Chloramine T conjugation method; theresulting specific activity is typically 1.5×10¹⁶ cpm/nmol. After 48hours, transfected cells are washed once with media (DMEM/F12 5% FBS).Non-specific binding sites are blocked by the addition of pre-warmedbinding media containing 5% non-fat dry milk and incubation at 37° C./5%CO₂ in a tissue culture incubator for one hour. The blocking media isdecanted and binding buffer containing ¹²⁵I anti-mIL-4R (clone M1; ratIgG1) is added to the cells and incubated with rocking at roomtemperature for 1 hour. After incubation of the cells with theradio-labeled antibody, cells are washed extensively with binding buffer(2×) and twice with phosphate-buffered saline (PBS). Cells are lysed in1 ml of 0.5M NaOH, and total radioactivity is measured with a gammacounter.

Using this assay, 293/EBNA co-transfected with DNAs encoding RANKdemonstrated transcriptional activation, as shown by detection ofmuIL-4R on the cell surface. Overexpression of RANK resulted intranscription of muIL-4R, as did triggering of the RANK by RANKL.Similar results are observed when RANK is triggered by agonisticantibodies.

Example 6

This example illustrates the association of RANK with TRAF proteins.Interaction of RANK with cytoplasmic TRAF proteins was demonstrated byco-immunoprecipitation assays essentially as described by Hsu et al.(Cell 84:299; 1996). Briefly, 293/EBNA cells were co-transfected withplasmids that direct the synthesis of RANK and epitope-tagged (FLAG®;SEQ ID NO:7) TRAF2 or TRAF3. Two days after transfection, surfaceproteins were labeled with biotin-ester, and cells were lysed in abuffer containing 0.5% NP-40. RANK and proteins associated with thisreceptor were immunoprecipitated with anti-RANK, washed extensively,resolved by electrophoretic separation on a 6-10% SDS polyacrylamide geland electrophoretically transferred to a nitrocellulose membrane forWestern blotting. The association of TRAF2 and TRAF3 proteins with RANKwas visualized by probing the membrane with an antibody thatspecifically recognizes the FLAG® epitope. TRAFs 2 and 3 did notimmunopreciptitate with anti-RANK in the absence of RANK expression.

Example 7

This example describes isolation of a ligand for RANK, referred to asRANKL, by direct expression cloning. The ligand was cloned essentiallyas described in U.S. Ser. No. 08/249,189, filed May 24, 1994 (therelevant disclosure of which is incorporated by reference herein), forCD40L. Briefly, a library was prepared from a clone of a mouse thymomacell line EL-4 (ATCC TIB 39), called EL-40.5, derived by sorting fivetimes with biotinylated CD40/Fc fusion protein in a FACS (fluorescenceactivated cell sorter). The cDNA library was made using standardmethodology; the plasmid DNA was isolated and transfected intosub-confluent CV1-EBNA cells using a DEAE-dextran method. Transfectantswere screened by slide autoradiography for expression of RANKL using atwo-step binding method with RANK/Fc fusion protein as prepared inExample 2 followed by radioiodinated goat anti-human IgG antibody.

A clone encoding a protein that specifically bound RANK was isolated andsequenced; the clone was referred to as 11H. An expression vectorcontaining murine RANKL sequence, designated pDC406:muRANK-L (in E. coliDH10B), was deposited with the American Type Culture Collection,Rockville, Md. (ATCC) on Dec. 20, 1996, under terms of the BudapestTreaty, and given accession number 98284. The nucleotide sequence andpredicted amino acid sequence of this clone are illustrated in SEQ IDNO:10. This clone did not contain an initiator methionine; additional,full-length clones were obtained from a 7B9 library (preparedsubstantially as described in U.S. Pat. No. 5,599,905, issued Feb. 4,1997); the 5′ region was found to be identical to that of human RANKL asshown in SEQ ID NO: 12, amino acids 1 through 22, except forsubstitution of a Gly for a Thr at residue 9.

This ligand is useful for assessing the ability of RANK to bind RANKL bya number of different assays. For example, transfected cells expressingRANKL can be used in a FACS assay (or similar assay) to evaluate theability of soluble RANK to bind RANKL. Moreover, soluble forms of RANKLcan be prepared and used in assays that are known in the art (i.e.,ELISA or BIAcore assays essentially as described in U.S. Ser. No.08/249,189, filed May 24, 1994). RANKL is also useful in affinitypurification of RANK, and as a reagent in methods to measure the levelsof RANK in a sample. Soluble RANKL is also useful in inducing NF-κBactivation and thus protecting cells that express RANK from apoptosis.

Example 8

This example describes the isolation of a human RANK ligand (RANKL)using a PCR-based technique. Murine RANK ligand-specific oligonucleotideprimers were used in PCR reactions using human cell line-derived firststrand cDNAs as templates. Primers corresponded to nucleotides 478-497and to the complement of nucleotides 858-878 of murine RANK ligand (SEQID NO:10). An amplified band approximately 400 bp in length from onereaction using the human epidermoid cell line KB (ATCC CCL-17) was gelpurified, and its nucleotide sequence determined; the sequence was 85%identical to the corresponding region of murine RANK ligand, confirmingthat the fragment was from human RANKL.

To obtain full-length human RANKL cDNAs, two human RANKL-specificoligonucleotides derived from the KB PCR product nucleotide sequencewere radiolabeled and used as hybridization probes to screen a human PBLcDNA library prepared in lambda gt10 (Stratagene, La Jolla, Calif.),substantially as described in U.S. Pat. No. 5,599,905, issued Feb. 4,1997. Several positive hybridizing plaques were identified and purified,their inserts subcloned into pBluescript SK⁻ (Stratagene, La Jolla,Calif.), and their nucleotide sequence determined. One isolate, PBL3,was found to encode most of the predicted human RANKL, but appeared tobe missing approximately 200 bp of 5′ coding region. A second isolate,PBL5 was found to encode much of the predicted human RANKL, includingthe entire 5′ end and an additional 200 bp of 5′ untranslated sequence.

The 5′ end of PBL5 and the 3′ end of PBL3 were ligated together to forma full length cDNA encoding human RANKL. The nucleotide and predictedamino acid sequence of the full-length human RANK ligand is shown in SEQID NO:12. Human RANK ligand shares 83% nucleotide and 84% amino acididentity with murine RANK ligand. A plasmid vector containing humanRANKL sequence, designated pBluescript:huRANK-L (in E. coli DH10B), wasdeposited with the American Type Culture Collection, Rockville, Md.(ATCC) on Mar. 11, 1997 under terms of the Budapest Treaty, and givenaccession number 98354.

Murine and human RANKL are Type 2 transmembrane proteins. Murine RANKLcontains a predicted 48 amino acid intracellular domain, 21 amino acidtransmembrane domain and 247 amino acid extracellular domain. HumanRANKL contains a predicted 47 amino acid intracellular domain, 21 aminoacid transmembrane domain and 249 amino acid extracellular domain.

Example 9

This example describes the chromosomal mapping of human RANK usingPCR-based mapping strategies. Initial human chromosomal assignments weremade using RANK and RANKL-specific PCR primers and a BIOS Somatic CellHybrid PCRable DNA kit from BIOS Laboratories (New Haven, Conn.),following the manufacturer's instructions. RANK mapped to humanchromosome 18; RANK ligand mapped to human chromosome 13. More detailedmapping was performed using a radiation hybrid mapping panel Genebridge4 Radiation Hybrid Panel (Research Genetics, Huntsville, Ala.; describedin Walter, M A et al., Nature Genetics 7:22-28, 1994). Data from thisanalysis was then submitted electronically to the MIT Radiation HybridMapper (URL: http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl)following the instructions contained therein. This analysis yieldedspecific genetic marker names which, when submitted electronically tothe NCBI Entrez browser (URL:http://www3.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=c&form=0),yielded the specific map locations. RANK mapped to chromosome 18q22.1,and RANKL mapped to chromosome 13q14.

Example 10

This example illustrates the preparation of monoclonal antibodiesagainst RANKL. Preparations of purified recombinant RANKL, for example,or transfixed cells expressing high levels of RANKL, are employed togenerate monoclonal antibodies against RANKL using conventionaltechniques, such as those disclosed in U.S. Pat. No. 4,411,993. DNAencoding RANKL can also be used as an immunogen, for example, asreviewed by Pardoll and Beckerleg in Immunity 3:165, 1995. Suchantibodies are likely to be useful in interfering with RANKL signaling(antagonistic or blocking antibodies), as components of diagnostic orresearch assays for RANKL or RANKL activity, or in affinity purificationof RANKL.

To immunize rodents, RANKL immunogen is emulsified in an adjuvant (suchas complete or incomplete Freund's adjuvant, alum, or another adjuvant,such as Ribi adjuvant R700 (Ribi, Hamilton, Mont.), and injected inamounts ranging from 10-100 μg subcutaneously into a selected rodent,for example, BALB/c mice or Lewis rats. DNA may be given intradermally(Raz et al., Proc. Natl. Acad. Sci. USA 91:9519, 1994) or intamuscularly(Wang et al., Proc. Natl. Acad. Sci. USA 90:4156, 1993); saline has beenfound to be a suitable diluent for DNA-based antigens. Ten days to threeweeks days later, the immunized animals are boosted with additionalimmunogen and periodically boosted thereafter on a weekly, biweekly orevery third week immunization schedule.

Serum samples are periodically taken by retro-orbital bleeding ortail-tip excision for testing by dot-blot assay (antibody sandwich),ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or othersuitable assays, including FACS analysis. Following detection of anappropriate antibody titer, positive animals are given an intravenousinjection of antigen in saline. Three to four days later, the animalsare sacrificed, splenocytes harvested, and fused to a murine myelomacell line (e.g., NS1 or preferably Ag 8.653 [ATCC CRL 1580]). Hybridomacell lines generated by this procedure are plated in multiple microtiterplates in a selective medium (for example, one containing hypoxanthine,aminopterin, and thymidine, or HAT) to inhibit proliferation ofnon-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocytehybrids.

Hybridoma clones thus generated can be screened by ELISA for reactivitywith RANKL, for example, by adaptations of the techniques disclosed byEngvall et al., Immunochem. 8:871 (1971) and in U.S. Pat. No. 4,703,004.A preferred screening technique is the antibody capture techniquedescribed by Beckman et al., J. Immunol. 144:4212 (1990). Positiveclones are then injected into the peritoneal cavities of syngeneicrodents to produce ascites containing high concentrations (>1 mg/ml) ofanti-RANK monoclonal antibody. The resulting monoclonal antibody can bepurified by ammonium sulfate precipitation followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can also be used, as canaffinity chromatography based upon binding to RANKL protein. Using themethods described herein to monitor the activity of the mAbs, bothblocking (i.e., antibodies that bind RANKL and inhibit binding to RANK)and non-blocking (i.e., antibodies that bind RANKL and do not inhibitbinding) are isolated.

Example 11

This example demonstrates that RANK expression can be up-regulated.Human peripheral blood T cells were purified by flow cytometry sortingor by negative selection using antibody coated beads, and activated withanti-CD3 (OKT3, Dako) coated plates or phytohemagglutinin in thepresence or absence of various cytokines, including Interleukin-4(IL-4), Transforming Growth Factor-β (TGF-β) and other commerciallyavailable cytokines (IL1-α, IL-2, IL-3, IL-6, IL-7, IL-8, IL-10, IL-12,IL-15, IFN-γ, TNF-α). Expression of RANK was evaluated by FACS in a timecourse experiment for day 2 to day 8, using a mouse monoclonal antibodymAb144 (prepared as described in Example 3), as shown in the tablebelow. Results are expressed as ‘+’ to ‘++++’ referring to the relativeincrease in intensity of staining with anti-RANK. Double labelingexperiments using both anti-RANK and anti-CD8 or anti-CD4 antibodieswere also performed.

TABLE 1 Upregulation of RANK by Cytokines Cytokine (concentration)Results: IL-4 (50 ng/ml) + TGF-β (5 ng/ml) + to ++ IL-4 (50 ng/ml) +TGF-β (5 ng/ml) ++++ IL1-α (10 ng/ml) − IL-2 (20 ng/ml) − IL-3 (25ng/ml) − IL-7 (20 ng/ml) − IL-8 (10 ng/ml) − IL-10 (50 ng/ml) − IL-12(10 ng/ml) − IL-15 (10 ng/ml) − IFN-γ (100 U/ml) −

Of the cytokines tested, IL-4 and TGF-B increased the level of RANKexpression on both CD8⁺ cytotoxic and CD4⁺ helper T cells from day 4 today 8. The combination of IL-4 and TGF-β acted synergistically toupregulate expression of this receptor on activated T cells. Thisparticular combination of cytokines is secreted by suppresser T cells,and is believed to be important in the generation of tolerance (reviewedin Mitchison and Sieper, Z Rheumatol. 54:141, 1995), implicating theinteraction of RANK in regulation of an immune response towards eithertolerance or induction of an active immune response.

Example 12

This example illustrates the influence of RANK.Fc and hRANKL onactivated T cell growth. The addition of TGFβ to anti-CD3 activatedhuman peripheral blood T lymphocytes induces proliferation arrest andultimately death of most lymphocytes within the first few days ofculture. We tested the effect of RANK:RANKL interactions on TGFβ-treatedT cells by adding RANK.Fc or soluble human RANKL to T cell cultures.Human peripheral blood T cells (7×10⁵ PBT) were cultured for six days onanti-CD3 (OKT3, 5 μg/ml) and anti-Flag (M1, 5 μg/ml) coated 24 wellplates in the presence of TGFβ (1 ng/ml) and IL-4 (10 ng/ml), with orwithout recombinant FLAG-tagged soluble hRANKL (1 μg/ml) or RANK.Fc (10μg/ml). Viable T cell recovery was determined by triplicate trypan bluecountings.

The addition of RANK.Fc significantly reduced the number of viable Tcells recovered after six days, whereas soluble RANKL greatly increasedthe recovery of viable T cells (FIG. 1). Thus, endogenous or exogenousRANKL enhances the number of viable T cells generated in the presence ofTGFβ. TGFβ, along with IL-4, has been implicated in immune responseregulation when secreted by the T_(H)3/regulatory T cell subset. These Tcells are believed to mediate bystander suppression of effector T cells.Accordingly, RANK and its ligand may act in an auto/paracrine fashion toinfluence T cell tolerance. Moreover, TGFβ is known to play a role inthe evasion of the immune system effected by certain pathogenic oropportunistic organisms. In addition to playing a role in thedevelopment of tolerance, RANK may also play a role in immune systemevasion by pathogens.

Example 13

This example illustrates the influence of the interaction of RANK onCD1a⁺ dendritic cells (DC). Functionally mature dendritic cells (DC)were generated in vitro from CD34⁺ bone marrow (BM) progenitors.Briefly, human BM cells from normal healthy volunteers were densityfractionated using Ficoll medium and CD34⁺ cells immunoaffinity isolatedusing an anti-CD34 matrix column (Ceprate, CellPro). The CD34⁺ BM cellswere then cultured in human GM-CSF (20 ng/ml), human IL-4 (20 ng/ml),human TNF-α (20 ng/ml), human CHO-derived Flt3L (FL; 100 ng/ml) in SuperMcCoy's medium supplemented with 10% fetal calf serum in a fullyhumidified 37° C. incubator (5% CO₂) for 14 days. CD1a⁺, HLA-DR⁺ DC werethen sorted using a FACStar Plus™, and used for biological evaluation ofRANK

On human CD1a⁺ DC derived from CD34⁺ bone marrow cells, only a subset(20-30%) of CD1a⁺ DC expressed RANK at the cell surface as assessed byflow cytometric analysis. However, addition of CD40L to the DC culturesresulted in RANK surface expression on the majority of CD1a⁺ DC. CD40Lhas been shown to activate DC by enhancing in vitro cluster formation,inducing DC morphological changes and upregulating HLA-DR, CD54, CD58,CD80 and CD86 expression

Addition of RANKL to DC cultures significantly increased the degree ofDC aggregation and cluster formation above control cultures, similar tothe effects seen with CD40L. Sorted human CD1a⁺ DC were cultured in acytokine cocktail (GM-CSF, IL-4, TNF-α and FL), in cocktail plus CD40L(1 μg/ml), in cocktail plus RANKL (1 μg/ml), or in cocktail plus heatinactivated (ΔH) RANKL (1 μg/ml) in 24-well flat bottomed culture platesin 1 ml culture media for 48-72 hours and then photographed using aninversion microscope. An increase in DC aggregation and clusterformation above control cultures was not evident when heat inactivatedRANKL was used, indicating that this effect was dependent onbiologically active protein. However, initial phenotypic analysis ofadhesion molecule expression indicated that RANKL-induced clustering wasnot due to increased levels of CD2, CD11a, CD54 or CD58.

The addition of RANKL to CD1a⁺ DC enhanced their allo-stimulatorycapacity in a mixed lymphocyte reaction (MLR) by at least 3- to 10-fold,comparable to CD40L-cultured DC (FIG. 2). Allogeneic T cells (1×10⁵)were incubated with varying numbers of irradiated (2000 rad) DC culturedas indicated above in 96-well round bottomed culture plates in 0.2 mlculture medium for four days. The cultures were pulsed with 0.5 mCi[³H]-thymidine for eight hours and the cells harvested onto glass fibersheets for counting on a gas phase β counter. The background counts foreither T cells or DC cultured alone were <100 cpm. Values represent themean±SD of triplicate cultures. Heat inactivated RANKL had no effect. DCallo-stimulatory activity was not further enhanced when RANKL and CD40Lwere used in combination, possibly due to DC functional capacity havingreached a maximal level with either cytokine alone. Neither RANKL norCD40L enhanced the in vitro growth of DC over the three day cultureperiod. Unlike CD40L, RANKL did not significantly increase the levels ofHLA-DR expression nor the expression of CD80 or CD86.

RANKL can enhance DC cluster formation and functional capacity withoutmodulating known molecules involved in cell adhesion (CD18, CD54),antigen presentation (HLA-DR) or costimulation (CD86), all of which areregulated by

CD40/CD40L signaling. The lack of an effect on the expression of thesemolecules suggests that RANKL may regulate DC function via an alternatepathway(s) distinct from CD40/CD40L. Given that CD40L regulates RANKsurface expression on in vitro-generated DC and that CD40L isupregulated on activated T cells during DC-T cell interactions, RANK andits ligand may form an important part of the activation cascade that isinduced during DC-mediated T cell expansion. Furthermore, culture of DCin RANKL results in decreased levels of CD1b/c expression, and increasedlevels of CD83. Both of these molecules are similarly modulated duringDC maturation by CD40L (Caux et al. J. Exp. Med. 180:1263; 1994),indicating that RANKL induces DC maturation.

Dendritic cells are referred to as “professional” antigen presentingcells, and have a high capacity for sensitizing MHC-restricted T cells.There is growing interest in using dendritic cells ex vivo as tumor orinfectious disease vaccine adjuvants (see, for example, Romani, et al.,J. Exp. Med., 180:83, 1994). Therefore, an agent such as RANKL thatinduces DC maturation and enhances the ability of dendritic cells tostimulate an immune response is likely to be useful in immunotherapy ofvarious diseases.

Example 14

This example describes the isolation of the murine homolog of RANK,referred to as muRANK. MuRANK was isolated by a combination ofcross-species PCR and colony hybridization. The conservation of Cysresidues in the Cys-rich pseudorepeats of the extracellular domains ofTNFR superfamily member proteins was exploited to design humanRANK-based PCR primers to be used on murine first strand cDNAs fromvarious sources. Both the sense upstream primer and the antisensedownstream primer were designed to have their 3′ ends terminate withinCys residues.

The upstream sense primer encoded nucleotides 272-295 of SEQ ID NO:5(region encoding amino acids 79-86); the downstream antisense primerencoded the complement of nucleotides 409-427 (region encoding aminoacids 124-130). Standard PCR reactions were set up and run, using theseprimers and first strand cDNAs from various murine cell line or tissuesources. Thirty reaction cycles of 94° C. for 30 seconds, 50° C. for 30seconds, and 72° C. for 20 seconds were run. PCR products were analyzedby electrophoresis, and specific bands were seen in several samples. Theband from one sample was gel purified and DNA sequencing revealed thatthe sequence between the primers was approximately 85% identical to thecorresponding human RANK nucleotide sequence.

A plasmid based cDNA library prepared from the murine fetal liverepithelium line FLE18 (one of the cell lines identified as positive inthe PCR screen) was screened for full-length RANK cDNAs using murineRANK-specific oligonucleotide probes derived from the murine RANKsequence determined from sequencing the PCR product. Two cDNAs, oneencoding the 5′ end and one encoding the 3′ end of full-length murineRANK (based on sequence comparison with the full-length human RANK) wererecombined to generate a full-length murine RANK cDNA. The nucleotideand amino acid seqeunce of muRANK are shown in SEQ ID Nos:14 and 15.

The cDNA encodes a predicted Type 1 transmembrane protein having 625amino acid residues, with a predicted 30 amino acid signal sequence, a184 amino acid extracellular domain, a 21 amino acid transmembranedomain, and a 390 amino acid cytoplasmic tail. The extracellular regionof muRANK displayed significant amino acid homology (69.7% identity,80.8% similarity) to huRANK. Those of skill in the art will recognizethat the actual cleavage site can be different from that predicted bycomputer; accordingly, the N-terminal of RANK may be from amino acid 25to amino acid 35.

Other members of the TNF receptor superfamily have a region of aminoacids between the transmembrane domain and the ligand binding domainthat is referred to as a ‘spacer’ region, which is not necessary forligand binding. In muRANK, the amino acids between 197 and 214 arepredicted to form such a spacer region. Accordingly, a soluble form ofRANK that terminates with an amino acid in this region is expected toretain the ability to bind a ligand for RANK in a specific manner.Preferred C-terminal amino acids for soluble RANK peptides are selectedfrom the group consisting of amino acids 214, and 197 of SEQ ID NO:14,although other amino acids in the spacer region may be utilized as aC-terminus.

Example 15

This example illustrates the preparation of several different solubleforms of RANK and RANKL. Standard techniques of restriction enzymecutting and ligation, in combination with PCR-based isolation offragments for which no convenient restriction sites existed, were used.When PCR was utilized, PCR products were sequenced to ascertain whetherany mutations had been introduced; no such mutations were found.

In addition to the huRANK/Fc described in Example 2, another RANK/Fcfusion protein was prepared by ligating DNA encoding amino acids 1-213of SEQ ID NO:6, to DNA encoding amino acids 3-232 of the Fc muteindescribed previously (SEQ ID NO:8).

A similar construct was prepared for murine RANK, ligating DNA encodingamino acids 1-213 of full-length murine RANK (SEQ ID NO:15) to DNAencoding amino acids 3-232 of the Fc mutein (SEQ ID NO:8).

A soluble, tagged, poly-His version of huRANKL was prepared by ligatingDNA encoding the leader peptide from the immunoglobulin kappa chain (SEQID NO:16) to DNA encoding a short version of the FLAG™ tag (SEQ IDNO:17), followed by codons encoding Gly Ser, then a poly-His tag (SEQ IDNO:18), followed by codons encoding Gly Thr Ser, and DNA encoding aminoacids 138-317 of SEQ ID NO:13. A soluble, poly-His tagged version ofmurine RANKL was prepared by ligating DNA encoding the CMV leader (SEQID NO:9) to codons encoding Arg Thr Ser, followed by DNA encodingpoly-His (SEQ ID NO:18) followed by DNA encoding amino acids 119-294 ofSEQ ID NO:11.

A soluble, oligomeric form of huRANKL was prepared by ligating DNAencoding the CMV leader (SEQ ID NO:9) to a codon encoding Asp followedby DNA ending a trimer-former “leucine” zipper (SEQ ID NO:19), then bycodons encoding Thr Arg Ser followed by amino acids 138-317 of SEQ IDNO:13.

These and other constructs are prepared by routine experimentation. Thevarious DNAs are then inserted into a suitable expression vector, andexpressed. Particularly preferred expression vectors are those which canbe used in mammalian cells. For example, pDC409 and pDC304, describedherein, are useful for transient expression. For stable transfection,the use of CHO cells is preferred; several useful vectors are describedin U.S. Ser. No. 08/785,150, now allowed, for example, one of the 2A5-3λ-derived expression vectors discussed therein.

Example 16

This example demonstrates that RANKL expression can be up-regulated onmurine T cells. Cells were obtained from mesenteric lymph nodes ofC57BL/6 mice, and activated with anti-CD3 coated plates, Concanavalin A(ConA) or phorbol myristate acetate in combination with ionomycin(anti-CD3: 500A2; Immunex Corporation, Seattle Wash.; ConA, PMA,ionomycin, Sigma, St. Louis, Mo.) substantially as described herein, andcultured from about 2 to 5 days. Expression of RANKL was evaluated in athree color analysis by FACS, using antibodies to the T cell markersCD4, CD8 and CD45RB, and RANK/Fc, prepared as described herein.

RANKL was not expressed on unstimulated murine T cells. T cellsstimulated with either anti-CD3, ConA, or PMA/ionomycin, showeddifferential expression of RANKL: CD4⁺/CD45RB^(Lo) and CD4⁺/CD45RB^(Hi)cells were positive for RANKL, but CD8⁺ cells were not. RANKL was notobserved on B cells, similar to results observed with human cells.

Example 17

This example illustrates the effects of murine RANKL on cellproliferation and activation. Various cells or cell lines representativeof cells that play a role in an immune response (murine spleen, thymusand lymphnode) were evaluated by culturing them under conditionspromoting their viability, in the presence or absence of RANKL. RANKLdid not stimulate any of the tested cells to proliferate. One cell line,a macrophage cell line referred to as RAW 264.7 (ATCC accession numberTIB 71) exhibited some signs of activation.

RAW cells constitutively produce small amounts of TNF-α. Incubation witheither human or murine RANKL enhanced production of TNF-α by these cellsin a dose dependent manner. The results were not due to contamination ofRANKL preparations with endotoxin, since boiling RANKL for 10 minutesabrogated TNF-α production, whereas a similar treatment of purifiedendotoxin (LPS) did not affect the ability of the LPS to stimulate TNF-αproduction. Despite the fact that RANKL activated the macrophage cellline RAW T64.7 for TNF-α production, neither human RANKL nor murineRANKL stimulated nitric oxide production by these cells.

Example 18

This example illustrates the effects of murine RANKL on growth anddevelopment of the thymus in fetal mice. Pregnant mice were injectedwith 1 mg of RANK/Fc or vehicle control protein (murine serum albumin;MSA) on days 13, 16 and 19 of gestation. After birth, the neonatescontinued to be injected with RANK/Fc intraperitoneally (IP) on a dailybasis, beginning at a dose of 1 μg, and doubling the dose about everyfour days, for a final dosage of 4 μg. Neonates were taken at days 1, 8and 15 post birth, their thymuses and spleens harvested and examined forsize, cellularity and phenotypic composition.

A slight reduction in thymic size at day 1 was observed in the neonatesborn to the female injected with RANK/Fc; a similar decrease in size wasnot observed in the control neonates. At day 8, thymic size andcellularity were reduced by about 50% in the RANK/Fc-treated animals ascompared to MSA treated mice. Phenotypic analysis demonstrated that therelative proportions of different T cell populations in the thymus werethe same in the RANK/Fc mice as the control mice, indicating that thedecreased cellularity was due to a global depression in the number ofthymic T cells as opposed to a decrease in a specific population(s). TheRANK/Fc-treated neonates were not significantly different from thecontrol neonates at day 15 with respect to either size, cellularity orphenotype of thymic cells. No significant differences were observed inspleen size, cellularity or composition at any of the time pointsevaluated. The difference in cellularity on day 8 and not on day 15 maysuggest that RANK/Fc may assert its effect early in thymic development.

Example 19

This example demonstrates that the C-terminal region of the cytoplasmicdomain of RANK is important for binding of several different TRAFproteins. RANK contains at least two recognizable PXQX(X)T motifs thatare likely TRAF docking sites. Accordingly, the importance of variousregions of the cytoplasmic domain of RANK for TRAF binding wasevaluated. A RANK/GST fusion protein was prepared substantially asdescribed in Smith and Johnson, Gene 67:31 (1988), and used in thepreparation of various truncations as described below.

Comparison of the nucleotide sequence of murine and human RANK indicatedthat there were several conserved regions that could be important forTRAF binding. Accordingly, a PCR-based technique was developed tofacilitate preparation of various C-terminal truncations that wouldretain the conserved regions. PCR primers were designed to introduce astop codon and restriction enzyme site at selected points, yielding thetruncations described in Table 2 below. Sequencing confirmed that noundesired mutations had been introduced in the constructs.

Radio-labeled (³⁵S-Met, Cys) TRAF proteins were prepared by in vitrotranslation using a commercially available reticulocyte lysate kitaccording to manufacturer's instructions (Promega). Truncated GST fusionproteins were purified substantially as described in Smith and Johnson(supra). Briefly, E. coli were transfected with an expression vectorencoding a fusion protein, and induced to express the protein. Thebacteria were lysed, insoluble material removed, and the fusion proteinisolated by precipitation with glutathione-coated beads (Sepahrose 4B,Pharmacia, Uppsala Sweden)

The beads were washed, and incubated with various radiolabeled TRAFproteins. After incubation and wash steps, the fusion protein/TRAFcomplexes were removed from the beads by boiling in 0.1%SDS+B-mercaptoethanol, and loaded onto 12% SDS gels (Novex). The gelswere subjected to autoradiography, and the presence or absence ofradiolabeled material recorded. The results are shown in Table 2 below.

TABLE 2 Binding of Various TRAF Proteins to the Cytoplasmic Domain ofRANK C terminal E206- E206- Truncations: S339 E206-Y421 M476 E206-G544Full length TRAF1 − − − − ++ TRAF2 − − − − ++ TRAF3 − − − − ++ TRAF4 − −− − − TRAF5 − − − − + TRAF6 − + + + ++

These results indicate that TRAF1, TRAF2, TRAF3, TRAF 5 and TRAF6 bindto the most distal portion of the RANK cytoplasmic domain (betweenamino-acid G544 and A616). TRAF6 also has a binding site between 5339and Y421. In this experiment, TRAF5 also bound the cytoplasmic domain ofRANK.

1. A cell line that produces a blocking antibody, wherein the blockingantibody binds to a human RANKL polypeptide as set forth in SEQ ID NO:13and inhibits the binding of the human RANKL polypeptide to a human RANKpolypeptide as shown in SEQ ID NO:6.
 2. A cell line that produces ablocking antibody, wherein the blocking antibody binds to a fragment ofa human RANKL polypeptide as shown in SEQ ID NO:13 and inhibits thebinding of the human RANKL polypeptide to a human RANK polypeptide asshown in SEQ ID NO:6, wherein the fragment is selected from the groupconsisting of a) an extracellular domain comprising amino acids 69-317of SEQ ID NO:13, and b) a receptor binding domain comprising amino acids162-317 of SEQ ID NO:13.